DESCRIPTION (Adapted from the application): Reduced nitric oxide (NO)-mediated vasodilatation in hypercholesterolemia is implicated in promoting atherosclerosis and its clinical manifestations. Oxidative stress associated with vascular disease plays an important role in interfering with the action of NO, but the precise cellular and molecular mechanisms by which NO mediates its actions, how oxidative stress interferes with NO, and how this relationship is altered in vascular diseases is unknown. The applicant's recent studies have shown that NO lowers intracellular Ca2+ concentration ([Ca2+]i) and relaxes vascular smooth muscle by accelerating Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum ATPase (SERCA) into intracellular stores, thereby rapidly lowering [Ca2+]i, and reducing store-operated Ca2+ influx. Preliminary studies indicate that in hypercholesterolemia, SERCA activity is reduced, impeding the ability of NO to lower [Ca2+]i, and relax vascular smooth muscle. Furthermore, these studies suggest that SERCA function is impaired due to oxidative stress arising from the reaction between NO and superoxide anion (O2-), resulting in peroxynitrite (OONO-)-mediated thiol oxidation, tyrosine nitration, and enzyme dysfunction. The hypothesis of the proposed studies is that oxidative stress in hypercholesterolemia results in molecular dysfunction of SERCA, interfering with NO action, causing impaired NO-mediated relaxation, and contributing to the pathogenesis of atherosclerosis. This hypothesis will be pursued in three aims to determine in hypercholesterolemic rabbits and in ApoE-deficient mice: 1) if dysfunction of SERCA is responsible for reduced effects of NO on [Ca2+]i; 2) if oxidative stress due to over-production of NO and O2- alters SERCA function by thiol oxidation, tyrosine nitration, or other post-translational modifications; and 3) if dysfunction of SERCA decreased responsiveness to NO, and atherosclerotic lesion is ameliorated in genetically modified Apo E-deficient mice that overexpress human SOD, or are deficient in gp91 phox, a subunit of NAD(P)H oxidase, or are deficient in the inducible isoform of nitric oxide synthase. These studies are designed to determine the molecular mechanisms by which oxidative stress in hypercholesterolemia impedes the cellular action of NO, and thereby further understand how impaired vasodilator function contributes to vascular disease.